| 1.Rat bone marrow mesenchymal stem cells isolation, cultivation, identification and neuronal conditioned medium induce it's differentiationObjective: To master the method of rat bone marrow mesenchymal stem cells primary cultivation, passage, and identification, to investigate use hippocampal neuron's conditioned medium to induce it differentiation.Methods: Use weight about (100-150) g SD rat, get the tibia and femur with germ free condition, expose the bone cavity, flush the cavity with antibiotic (penicillion 100 u/mL, streptomycin 100u/mL) DMEM medium, collect it in centrifuge tube, susupension it and cultured with 10% FBS medium into the cultured bottle, incubate in 37℃,5% CO2 incubator. 3d change the half medium, 5 d change the medium completely, then change the medium every 3 d. use CD34 and CD44 as the primary antibody to do the immunocytochemistry staining, PBS as the negative control, add the biotinize secondary antibody, 37℃, 1 h, PBS wash 3 times, add horseradish peroxidase, staining with DAB, stop with water, ethanol dehydration, xylene transparent, fix it with gum, observe under microscope. Use primary cultured hippocampal neuron's medium as conditioned medium, b-FGF group add 10% DMSO, b-FGF and L-DMEM, the last group use Neurobasal plus B27 as serum free medium, negative control is DMEM medium(with 10%FBS). P5 BMSCs planted on the cover slip wih covering 0.1% PLL ( Poly-L-Lysine ) in 6 wells plate, when the BMSCs growth on the cover slip and change with Neurobasal medium, hippocampal conditioned medium and serumfree medium, induced the cell for 12 h and 1 d, after that fixed with paraformaldehyde. BMSCs following induce with different medium, use immunocytochemistry by MAP-2, NSE and GFAP antibody, observe the positive expression of MAP-2, NSE and GFAP, count the positive ratio of different groups and photographed.Results: BMSCs primary culture for 1 d, few of them adhere to the bottom, 2 d cleavage and proliferate, show oval ship or multiform, often form cell cluster, 4 d prolife rapidally, 3 d change the medium half, the impurity is clear, the cell morphology is obvious, the body is larger and prolife. 7-9 d is full on the bottom, about 95% emerge. BMSCs after passage growth fast,4h is adhere to the substance, 24h adhere completely and prolife. 5-7d can growth about 90% of the bottom. After 1'st passage, the purity is about 95%, passage it for 6 generation, each passage is the same in morphology and growth. BMSCs H-E staining show spindle and multiform, the nuclei is large, round and heavy dye, basophilic, cytoplasma is light, acidophilic, cell paralled. Rabbit anti rat CD34 show negative and colorless, CD44 cell adhere molecular positive, nuclei and cytoplasma show brown. Hippocampal neuron conditioned medium, b-FGF medium and B27 plus neurobasal (serum free) medium induced the BMSCs for 12 h and 1 d, after inducing the cell body changed into round and pyramid gradually, process elongate, 1 d some of them can connect each other. With immunocytochemistry stainin, 12 h and 1 d labeled with MAP-2, NSE and GFAP, count the positive showed conditioned medium induced BMSCs positive ratio is the highest, express NSE, MAP-2, GFAP higher than others, have the significant statistically difference(P<0.05)Conclusions: 1.Full bone marrow culture method and adhere way is a successful method to isolation, purify the BMSCs in vitro, and it can prolife rapidly and stable. 2. BMSCs in vitro with immunocytochemistry staining, it's express mesenchymal stem cells characteristic, not the hemotopoitic stem cells. 3. With b-FGF DMEM medium, hippocampal conditioned medium, serum free medium induced BMSCs 12 h and 1 d, BMSCs can differentiate into neuronal like cell and glial like cell, with immunocytochemistry identification it's express NSE, MAP-2 and GFAP positive characteristic. 3. With b-FGF DMEM medium, hippocampal medium, serum free medium induce BMSCs, 12 h is the highest ratio, it's about 44.78%, conditioned medium compare with others, there are statistically differences.2. CVS model following SAH and ethological, biological detectionObjective: Observe rat autologous blood cisterna magna injection to produce CVS model following SAH, detect blood and brain NOS and ET-1 content varation, the diameter of cerebro vessel, value the model of CVS from ethological performance.Methods: Use double hemorrhage cistern magna injection method to produce SAH model, separate the atlanto-occipital membrane and cut anterio the end of tail about 5-6 cm, get the arterial blood 0.2-0.3 mL from tail artery, with stereotaxica coordinate inject the arterial blood from atlanto-occipital membrane about 5 min, and 2 d later inject the blood again the same way. Rat was valued with neurological functional score, in 1 d, 2 d, 3 d, 5 d, 7 d compare SAH and control group. After vessel perfusion with 10% of india ink detect the diameter of cerebral artery with magnification of 45, with Nikon NIS BR 3.0 and Image J analysis system, detect the basal artery, introcarotid artery, and middle cerebral artery diameter. With the same perfusion way, filling the microvessel, 1 mm thick slice around the hippocampus 4 mm posterior bregma, valure the vessel filling under 45 magnification. SAH 1 d, 2 d, 3 d, 5 d, 7 d extract the eyeball, get 2 mL blood, get the hippocampus and cortex of brain, use serum and detect the ET-1 and NOS according to it's manual, after injection 14 d, do Morris mazer test. All data process with SPSS14.0 soft ware and use one way annova, compare control and experimental group with Dunnett-t method, among groups with SNK-q test, with neurological functional score and cerebral diameter, NOS and ET-1 use multiple linear regression, Morris mazer mean escape time and NOS, ET-1, cerebral diameter also use multiple linear regression. Results: after SAH rat show decrese in eating, some kind of abnormal ethology, show lower reaction, lower alartness, sleepy, bloom, lack of diat, it can be better on 2'nd day, but double hemorrhage aggrevate, some of them can be paralysis. After injection there are blood clot in cerebullar medullary cistern, 1 d can see obsolete blood. Posterior 4 mm of bregma cortex vessel attenuate and lessen, the branches to medullary is lessen, but the control group show normal and abundant. With image analysis detect the vessel, SAH 2 d and 3 d the diameter have statistically difference versus control group. For neurological function score, SAH 1 d, 2 d, 3 d, 5 d have higher score, versus control group have significant difference. SAH 1 d, 2 d, 3 d, 5 d, 7 d blood and brain ET-1 were higher than sham. With multiple linear regression, neurological function score is related with ET-1 and middle cerebral artery. The average swimming speed is the same, while the swimming distance and escaping time of SAH group is longer than others, SAH span the flat less than control group.Conclusions: 1. Rat autologous tail blood cistern magna injection double hemorrhage model, there are clot around the sulcus and gyrus, with microperfusion shows clearly brain vessel hypoperfusion. 2. Compare with control group, SAH rat diameter of BA, MCA, ICA is thinning, especially for MCA. 3. SAH brain and blood ET-1, NOS show NOS lower than control group, ET-1 higher than the others. 4. SAH neurological score higher than others. 5. Morris mazer average escaping time of SAH is longer than control group, span times lower than sham.3. BrdU Labelled BMSCs transplant to CVS rat following SAH from lateral ventricle injectionObjective: To transplant labeled BMSCs in to SAH following CVS from lateral ventricle, investigate the labeled cell development in brain, valure the cell transplantation effect with ethological method. Methods: P5 BMSCs, with BrdU to labelled for 2 d, after that check it by immnunocytochemistry staining with BrdU antibody. Then use labelled cell suspension, as 5×106 cell density to transplant to rat brain from lateral ventricle. By neurological score valure the 3 groups: SAH group, BMSCs group, and only transplant DMEM group. Sandomize 3 group, SAH didn't transplant cell, DMEM only inject 30μL DMEM medium after 2'nd blood injecton 2 d, BMSCs group after SAH 2 d under stereotaxic coordinate inject 30μL BMSCs suspension as previous describe density. The Morris mazer and ethological exam after 14 d. Under stereotaxic coordinate, rat lateral ventricle localize is: posterior to bragma 1.2 mm, median line 2 mm, and the deep is 4.5 mm, after injection 14 d, fixed with paraformadehyde and get the brain slices, by immunohistochemistry staining method to show the positive BrdU labeled BMSCs. Detect neurological score at 1 d, 2 d, 3 d, 5 d, 7 d after SAH inject DMEM and BMSCs, 14 d do the Morris mazer experiment.Results: BrdU labelled BMSCs, immnunohistochemistry staining show nuclei brown is positive, negative is baby blue. Localize lateral ventricle under stereotaxic coordinate with bragam, it's accurate into lateral ventricle and have no other untoward reaction, compare with DMEM group, BMSCs group sensory and motor disable no aggravate. With DAB staining of brain slice, the positive cell of BrdU labelled show brown color, positive cell irregular line within the negative cells. Neurological score show 1 d is the highest, with time it's decrease little by little. DMEM group and SAH group have no statistically difference, Morris mazer BMSCs escaping time is shorter than SAH and DMEM group, spaning flat times show BMSCs more than others, have statistically difference.Conclusions: 1. BrdU can labelled BMSCs, with BrdU antibody immnunocytochemistry staining show brow color. 2. Under stereotaxic coordinate localize posterior bragma 1.2 mm, median line 2 mm, depth 4.5 mm can accurate inject into lateral ventricle, have no other side effect. 3.Labelled BrdU BMSCs, show positive BrdU cell in the brain slice. 4. Rat neurological score show BMSCs group 7 d has recover in motor and sensory in certain degree. 5. Morris mazer show BMSCs group have some kind of recover in learning and memory.4. The apoptosis investigation of hippocampus neuron after transplantation of BMSCs to CVS rat following SAHObjective: after transplantation BMSCs to CVS rat following SAH, observe the expression of Bcl-2 and Bax protein which are related with apoptosis in brain and hippocampus.Methods: With the SAH group, DMEM group and BMSCs group brain slices, use rabbit anti rat Bcl-2 and Bax as primary antibody, immunohistobiochemistry staining method to observe the morphological change. With Image analysis soft ware count the positive cell ratio of each groups. Get the SAH, DMEM and BMSCs group rat brain in 14 d, dissect the hippocampus, add the lysate about 1 mL per 100 mg brain tissue, ultrasonic clearage and centrifuge hypothermia, get the supernatant, detect it's protein content with Coomassie agent. Protein denaturation and spotting, electrophoresis and transfer to nitrocellulose membrane. Blocking with defatted milk powder, add primary antibody of Bcl-2 and Bax, 4℃over night, add horseradish peroxidase, immune-developing, with Hema analysis system to detect each group protein expression level.Results: Compare with SAH, BMSCs at hippocampal slice Bcl-2 positive expression increase, Bax expression decrease, SAH group at the hippocampal slice Bcl-2 decrease and Bax increase. DMEM group Bcl-2 decrease and Bax increase. Western blot show, control group, SAH group, BMSCs group GAPDH expression is the same, BMSCs group Bcl-2 is higher than the others, and Bax expression is lower. Have statistically difference.Conclusions: 1. SAH following CVS transplant BMSCs, the express of inhibit apoptosis gene is decrease and promote apoptosis gene is increase. 2. SAH 14 d rat brain promote apoptosis gene expression increase and inhibit apoptosis gene decrease. 3.There are a little expression of promote apoptosis gene expression in DMEM group.5. The blood NOS, ET-1 content change and micro-ultral structure change after BMSCs transplantation to CVS rat following SAHObjective: To investigate blood NOS, ET-1 changes of BMSCs transplantation to CVS rat following SAH, transmission electron microscope observe the cerebrovascular and neuron's ultra-micro-structural change.Methods: Animal grouping, SAH model and cell transplantation as previous subscribe, collect blood after BMSCs transplantation 14 d, detect the content of NOS and ET-1, as previous. Get the blood at 1 d, 3 d, and 7 d. Detect the NOS and ET-1 content in blood, as previous describe. Get the brain cortex posterior bregma about 1mm×1mm×2mm, fix it with 2.5% glutaral phosphates about 24h, ultral thin slices for TEM, wash it with 0.1mol/L phosphate 3 times, 1% osmic acid fix, ethanol and acetone dehydration, bed with epoxy resins, polymerization, 0.5μm thickness slicing the tissue, 1% toluidin blue-azure thickness about 50 nm, dredge with copper screen, uranyl acetate and citric acid staining, Hitachi H-7500 TEM observe ultra-structure of the brain cortex neuron and microvascular, photography. Comparasion between groups use one way ANOVA, among groups use SNK-q test, P<0.05 represent difference.Results: Compare with control group, SAH 1 d, 3 d and 7 d blood NOS decrease and ET-1 increase, as SAH 3 d increase for typical, have statistically difference. BMSCs group blood ET-1 decerease versus SAH group, NOS increase, have statistically difference. Compare with control group, BMSCs group ET-1 is still in high level, have statistically difference, NOS have certain level decrease, but have no statistically difference. TEM ultra-micro-structure show control group neuclus is big and round, Chromosome density is even, karyosome is clear, nuclear membrane is intergreted. Endothelial cell endomembrane is smooth, cell neuclear, apparatus, tight injunction is clear, endothalia matrix and basal lamina is integrated, stratification is clear, vessel have no narrow. SAH group cortex neuron and microvascular endothelial edema is the highest, mitochondria swelling, most of crest and membrane emerge, disappear, some of them medulla change, there are vaculous in cytoplasma, neuron nuclear chromosome concentrate, heterochromatin increase, mitochnodia, rough endoplasm reticulum swelling, even collapse. BMSCs transplantation can see little bit of edema in cortex neuron and microvasular endothelial, pinocytosis bulb attenuate in vascular endothelial cell.Conclusions: BMSCs lateral ventricle transplantation to SAH following CVS rat blood ET-1 decreas, NOS increase, SAH group neuron necrosis deeply and some of microvessel stenosis, after transplant BMSCs is attenuate the effect of CVS, imply cerebral vascular have a certain degree of release.
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